Diagnostic value of PPARδ and miRNA-17 expression levels in patients with non-small cell lung cancer

The PPARδ gene codes protein that belongs to the peroxisome proliferator-activated receptor (PPAR) family engaged in a variety of biological processes, including carcinogenesis. Specific biological and clinical roles of PPARδ in non-small cell lung cancer (NSCLC) is not fully explained. The association of PPARα with miRNA regulators (e.g. miRNA-17) has been documented, suggesting the existence of a functional relationship of all PPARs with epigenetic regulation. The aim of the study was to determine the PPARδ and miR-17 expression profiles in NSCLC and to assess their diagnostic value in lung carcinogenesis. PPARδ and miR-17 expressions was assessed by qPCR in NSCLC tissue samples (n = 26) and corresponding macroscopically unchanged lung tissue samples adjacent to the primary lesions served as control (n = 26). PPARδ and miR-17 expression were significantly lower in NSCLC than in the control (p = 0.0001 and p = 0.0178; respectively). A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential in discriminating NSCLC from the control with an area under the curve (AUC) of 0.914 for PPARδ and 0.692 for miR-17. Significant increase in PPARδ expression in the control for current smokers vs. former smokers (p = 0.0200) and increase in miR-17 expression in control tissue adjacent to adenocarcinoma subtype (p = 0.0422) were observed. Overexpression of miR-17 was observed at an early stage of lung carcinogenesis, which may suggest that it acts as a putative oncomiR. PPARδ and miR-17 may be markers differentiating tumour tissue from surgical margin and miR-17 may have diagnostic role in NSCLC histotypes differentiation.


Materials and methods
Patients and tissue collection. Twenty six (26) adult patients with NSCLC were qualified for the study- For analysis, lung tissue samples (approximately 100 mg) were collected from primary lesion and surgery margin (2 cm away from the primary lesion), as a control group (macroscopically unchanged lung tissue). The resected primary tumours were post-operatively subjected to the histopathological analysis. NSCLC samples were classified as: squamous cell carcinoma (SCC) and adenocarcinoma (AC). All cases were primary tumours without chemo-or radiotherapeutic treatment. RNA isolation, qualitative and quantitative RNA evaluation. Lung tissue samples were placed in fixRNA buffer (Eurx, Gdańsk, Poland), then divided into smaller parts, homogenized and frozen at -80 °C until use. Isolation of total RNA from tissue homogenates was performed using the mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. Qualitative and quantitative evaluation of the isolated RNA was performed by spectrophotometric method by measuring the absorbance with the Eppendorf BioPhotometerTM Plus apparatus (Eppendorf, Hamburg, Germany), at 260/280 nm wavelengths. Prepared RNA was divided into portions and frozen at -80 °C until the real-time polymerase chain reaction (qPCR) was performed.
Evaluation of gene/miRNA expression. The reverse transcription (RT) reaction for genes was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), in a volume of 20 μl. The reaction mixture contained: 10 × RT buffer, 25 × dNTP Mix (100 mM), 10 × RT Random Primers, MultiScribe™ Reverse Transcriptase (50 U/µL), RNase Inhibitor and nuclease-free water. 100 ng of total RNA was added to the reaction mixture. The negative control in the RT reaction was carried out using water instead of RNA. The following RT reaction conditions were used: 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C and cooling at 4 °C. The reverse transcription (RT) for miRNA of 5 μl (10 ng) of total RNA in a 15-μl reaction was carried out using a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). RT master mix contained: 25 × dNTP Mix (100 mM), MultiScribe™ Reverse Transcriptase (50 U/µL), 10 × RT buffer, RNase Inhibitor (20 U/µL), nuclease-free water and the specific RT primers (small RNA-specific RT primers) included in individual TaqMan® MicroRNA Assays: hsa-miR-17-5p (CAA AGU GCU UAC AGU GCA GGUAG) and RNU6B (CGC AAG GAT GAC ACG CAA ATT CGT GAA GCG TTC CAT ATT TTT ) as endogenous control (Applied Biosystems, Carlsbad, CA). RT reaction was performed in a Personal Thermocycler (Eppendorf, Germany) in the www.nature.com/scientificreports/ following conditions: 30 min at 16 °C, followed by 30 min at 42 °C, then the samples were heated to 85 °C for 5 min, and held at 4 °C. RT products were stored at -20° C until further analysis. Relative gene/miRNA expression was assessed by real-time polymerase chain reaction (qPCR) using a 7900HT Fast Real-Time PCR System apparatus (Applied Biosystems, Carlsbad, CA). A total reaction mixture volume of 20 μl contained: cDNA (1-100 ng), KAPA PROBE FAST qPCR Master Mix (2X) ABI Prism™ (Kapa Biosystems Ltd, London, UK), RNase-free water and 20xTaqMan® Gene Expression Assay for the following genes: PPARδ (Hs00987008_m1), and ACTB (Hs99999903_m1) selected as the reference gene in the qPCR reaction. Assays for the following miRNA: miRNA-17, and RNU6B (endogenous control) were used in the qPCR reaction. The relative expression levels of the analyzed gene/miRNA were evaluated by the delta-delta CT method (TaqMan Relative Quantification Assay software, Applied Biosystems) and presented as RQ values relative to the ACTB/ RNU6B reference gene/miRNA, respectively. The following formula was used to determine the ΔΔCT value: ΔΔCT = ΔCT test sample-ΔCT calibrator sample. For calibrator (commercial sample-Human Lung Total RNA, Invitrogen™), RQ value was considered equal to 1. The obtained results were compared in terms of the NSCLC histopathological subtype, cancer stage (TNM, AJCC), age of patients, gender and smoking history. In the case of the test samples, the increased expression was recognized when the RQ value was more than 1 and the decreased expression-when the RQ value was less than 1.
Statistical analysis. Statistical analysis was performed using Statistica for Windows 10.0 software (StatSoft, Cracow, Poland) (v.13). In order to check the occurrence of statistical significance between the analyzed groups, Mann-Whitney U-test and was used. The Spearman rank correlation coefficient was used to measure the direction and strength of the relationship for individual variables. To assess the specificity and sensitivity of PPARδ and miR-17 as potential diagnostic predictors that could differentiate NSCLC from the operating margin, the receiver operating characteristic (ROC) curve was analyzed and the area under the curve (AUC) was resolved with a 95% confidence interval (CI). The results of relative expression analysis (RQ value) are presented as median values. Statistical significance was determined at p < 0.05. Informed consent. Informed consent was obtained from all patients included in the study.

Results
Relative gene/miRNA expression in NSCLC tissue vs. controls. The relative expression level of PPARδ and miR-17 in NSCLC tissues vs. control tissues was compared. The obtained results showed a decreased expression of PPARδ in 81% of NSCLC samples and its increased expression in 88% of control samples. The RQ value of miR-17 was increased in all samples, both NSCLCs and controls. Statistically significant difference in the relative expression of PPARδ and miR-17 between the NSCLC samples and the control tissue (p = 0.0001 and p = 0.0178; respectively, Mann-Whitney U test) was observed. The obtained results are presented in Fig. 1a and Table 2.
We also evaluated the potential of the PPARδ and miR-17 as diagnostic classifiers for NSCLC by performing receiver operating characteristic curves and area under the curve (ROC-AUC) analyses. The obtained results revealed that PPARδ and miR-17 expressions were able to differentiate macroscopically unchanged lung tissue from NSCLC with AUC-ROC values of 0.914 (95% CI: 0.840-0.989; p = 0.00001) and 0.692 (95% CI: 0.840-0.989, p = 0.0117), respectively. Both the specificity and sensitivity of PPARδ in tissue type prediction were 84.6%, while the specificity of miR-17 was 88.5% and its sensitivity was 50% (see Fig. 1b).

Gene/miRNA RQ values vs. clinicopathological parameters. The obtained RQ values for PPARδ
gene and miR-17 were analyzed in relation to clinical features of patients: age at time of diagnosis, gender and smoking history as well as histopathological characteristics of tumours (according to pTNM and AJCC classifications and NSCLC subtypes). Table 3a, b indicates RQ values (medians) of the studied gene/miRNA in relation to the mentioned clinicopathological parameters.
There was no significant correlation between RQ values of gene/miRNA and patients' age (two age groups: ≤ 65 years and > 65 years) and gender (p > 0.05; Mann-Whitney U test). Differences in the level of expression of the PPARδ gene depending on the history of cigarette smoking (tobacco addiction and consumption) were observed, but only in the control group. A significant increase in the RQ value of PPARδ in the control tissue was demonstrated in the case of patients who were still smoking (current smokers) vs. those who stopped smoking (former smokers) (p = 0.0200; Mann-Whitney U test). We did not observe such a relationship for miR-17 (p > 0.05; Mann-Whitney U test). In NSCLC group, the level of gene/miRNA expression was lower among current smokers compared to the patients who stopped smoking, but without statistical significance (p > 0.05; Mann-Whitney U test).
No significant differences in RQ values PPARδ gene and miR-17 depending on the number of cigarettes smoked out, presented in pack years (≤ 40 vs. > 40PY) was showed (p > 0.05; Mann-Whitney U test). In addition, an analysis was carried out in the entire group of smokers to see if there is a correlation between PPARδ/ miRNA-17 expression and the amount of cigarettes smoked in relation to the length of the smoking (PYs).   . 2). There were no statistically significant differences in the level of PPARδ and miR-17 depending on tumour staging according to pTNM and AJCC classifications (p > 0.05; Mann-Whitney U test).
Correlation between the expression level of PPARδ gene and miRNA-17. The simultaneous decreased expression level of PPARδ and increased expression level of miRNA-17 were observed in 21 NSCLC tissue samples (81%) and in 3 control tissue samples (12%). There were no statistically significant correlations between the expression of studied PPARδ and miR-17 neither in the NSCLC nor in tumour tissue margins (p > 0.05; Spearman's rank correlation coefficient) (see Fig. 3).

Discussion
Peroxisome proliferator-activated receptors, PPARs, are transcription factors whose main role is to control fatty acid metabolism and maintain glucose homeostasis, as well as to participate in cell proliferation and differentiation. To date, three PPAR isotypes have been identified: α, β/δ and γ. Each of them is a product of a separate gene, has a different expression profile in tissues, is activated by specific ligands and is involved in various, although often complementary, cellular processes. The most common isotype is PPARβ/δ expressed in all tissues at a similar level. So far, it has been demonstrated that it is involved in the control of energy homeostasis and www.nature.com/scientificreports/ thermogenesis. It is also involved in the regulation of fatty acid β-oxidation and cholesterol transport, which is why its ligands are proposed as drugs in the treatment of metabolic syndrome X. In addition, it plays an important role in the proliferation of keratinocytes and the process of wound healing 4 . In contrast to these roles of PPARβ/δ established in normal physiology, the effect of PPARβ/δ in carcinogenesis remains still controversial. Previous studies have confirmed the relationship of PPARβ/δ with lung cancer 2,10,29 , but the mechanism of its effects has not been determined. The aim of our study was to determine the level of the PPARδ gene expression in patients with NSCLC. The control material was a fragment of the macroscopically unchanged lung tissue surrounding the tumour from the same patient. To the best of our knowledge, we were the first to observe significant differences between the expression of PPARδ in the lung tumour and control tissue. In addition, we confirmed the high sensitivity and specificity of the PPARδ potential for prediction of type of tissue. Such a difference indicate the possible utility of PPARδ gene expression level as a diagnostic marker. Literature data describing expression of PPARβ/δ mRNA and/or PPARβ/δ protein in both types of tissues are inconclusive and largely dependent on the method used to determine its expression patterns 9 . Immunohistochemistry showed higher PPARβ/δ expression in tumours than www.nature.com/scientificreports/ in nontransformed control lung tissue, but Western blot analysis did not support this concept 9,30 . On the other hand, tissue microarray indicated moderate expression of PPARβ/δ in respiratory epithelial cells of the bronchus and lack or weaked expression in human lung cells carcinomas 9 . Those results are consistent with ours and the qPCR technique that we used is considered the "gold standard" in microarray validation 31 , thus supporting the diagnostic significance of PPARδ. The observed decreased PPARδ expression in NSCLC and its increased level in adjacent normal lung tissue may indicate its putative role as tumour suppressor gene. Literature data indicate that PPAR receptors, may be the target of anti-cancer therapy in intestine, mammary gland, prostate and lymphatic system cancers, because they exert antiproliferative effects 4 . The suppressive role of PPARβ/δ in the lung cancer was also signaled by Peters et al. 9 who claimed that in vast majority of human lung cancers (both SCC and AC) expression of PPARβ/δ was low or none. Other research, conducted on PPARβ/δ ligand, L165041 has proved that it inhibits human lung adenocarcinoma cell proliferation 29 , while the study performed on transgenic mouse model has showed that lack of PPARβ/δ expression is associated with exacerbation of lung cancer 32 .
On the other hand, the prooncogenic properties of PPARδ have also been observed. PPARδ gene upregulation in 3 rd stage of cancer and its impact on the metastasis development in various cancer models (lung, breast, colorectal) in vivo was reported 17 . Genini et al. 2 have observed increased PPARβ/δ mRNA level in NSCLC comparing to the normal lung tissue and concomitantly up-regulated VEGF and components of the Cox-2/prostaglandin synthetic pathway in a subset of NSCLC, thus suggesting that activation of these pathways plays a role in lung carcinogenesis. Also on protein level the increased PPARδ expression was demonstrated in the subtypes of lung cancer, i.e., adenocarcinoma and squamous carcinoma 30 . Regarding receptors ligands, Han et al. 33 have showed that PPARβ/δ agonist (GW501516) stimulates human lung carcinoma cell proliferation. Summarizing the above, PPARδ is able to modulate both cancer cells and unaltered cells in the surroundings of tumour depending on its influence on target genes 10 . Ligand-bound PPARβ/δ induces expression of target genes, while if it is not bound by its ligands it can repress the transcription of its target genes. PPARβ/δ activity may also be affected by the presence or absence of cofactors and repressors 10,14 . It is suggested that PPARδ plays different roles depending on the site of its expression: in normal cells in the tumour microenvironment it causes promotion of tumourigenesis, while in cancer cells -its suppression 34,35 . It seems to be consistent with the findings of our research. www.nature.com/scientificreports/ Considering the relationship between the status of PPARδ expression and the clinical and pathological features of NSCLS, in our research we didn't observe any differences in PPARδ expression depending on the gender or age of the subjects, the number of cigarettes smoked out presented in pack years, the stage of the cancer according to TNM and AJCC classifications, as well as the histological subtype of the cancer (SCC vs. AC). To our knowledge, there are no published studies comparing the expression of PPARδ with the above mentioned features, apart from Pedchenko et al. 30 who, with the use of the immunochemical method, also didn't notice any significant correlation between the level of PPARδ and history of smoking, tumour stage or tumour histology.
However, we observed significantly higher PPARδ expression in unchanged lung tissue of current smokers comparing to former smokers. This result seems to confirm the PPARβ/δ activation mechanism proposed by Sun et al. 1 who showed a time-and dose-dependent induction of PPARβ/δ protein by nicotine through nicotinic acetylcholine receptor nAChR-mediated activation of PI3K/mTOR pathway. The role of PPARβ/δ in mediating the effect of nicotine on the growth of cancer cells is simultaneously suggested 1 . In our study, such a role of PPARδ is visible already at an early stage of carcinogenesis and may be the result of the genotoxic action of oxidative stress which leads to early molecular changes. We did not find any other research comparing expression of PPARδ depending on smoking status.
Regarding miR-17, the literature data confirm that miR-17-92 cluster encoding miR-17-5p and miR-17-3p is necessary for normal lung development and alterations in its expression have been reported in various pulmonary diseases, such as lung cancer 25,36 . High expression of miR-17 in lung cancerous tissue was observed by Saito et al. 37 . Similarly, Chen et al. 21 noted elevated expression levels in tumour tissue and also in serum of patients with lung cancer. Our results confirmed overexpression of miR-17 in all NSCLC and control tissues samples, however, with significantly lower median RQ value in NSCLC. A 15-fold increase in miR-17 expression in NSCLC and a 30-fold increase in miR-17 expression in tissue surrounding the tumour seems interesting and speaks for an oncogenic role of miR-17. Current publications, however, are not conclusive as to the functional mechanism of the miR-17 and the results are inconsistent. Cloonan et al. 38 have shown that miR-17-5p may be either an oncogene or a tumour suppressor gene in different cell environments, depending on the expression of other transcriptional regulators. MiR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes, both pro-and anti-proliferative, involved in the transition between these phases [38][39][40] .
In our study, we observed differences between the expression of miR-17 and the stage of cancer according to the TNM and AJCC classification, showing its higher expression in more advanced stages of cancer, however those results were statistically insignificant. We noted that T2 and T3 tissue samples were characterized by a comparable expression of miR17, higher than T1. Increased expression of miR-17 was observed in patients with stage II and III cancer, and this elevated level increased with the presence of histologically confirmed lymph node metastasis: higher expression was noted in patients with N1 + N2 feature. This result is in accordance with oncogenic properties of the miR-17, belonging to miR-17-92 cluster, which overexpression promotes cell proliferation and progression of various cancers including NSCLC [41][42][43][44] . The degree of miR-17-5p overexpression correlated with lung cancer aggressiveness, metastasis status in the lung cancer patients and responsiveness to chemotherapeutics 42,45 . A possible mechanism by which miR-17 is involved in carcinogenesis as the classical oncogene is an enhancement of cell proliferation through modulation of the PI3K/Akt/mTOR pathway 46 .
However, there are also reports supporting the suppressor role of miR-17. For example, downregulation of miRNA-17-5p in NSCLC tissues and cell lines comparing to the healthy controls was noted 25 ; lower miR-17-5p expression was also observed in lung adenocarcinoma initiating cells 47 . However, miR-17-5p may play different roles at different stages of lung cancer 25 which might explain, to some extent, the discrepant results. Additionally, the profile of miR-17 expression appears to be determined by the biological material which is analyzed. For instance, in serum mir-17 expression pattern does not reflect the pattern observed in lung tissue. Similarly to our results, the lack of statistical significance of miR-17 expression between I and II-IV stage of NSCLC according to AJCC classification was observed by Qi et al. 48 , but increased expression referred to the early stages of tumour development. Moreover, significantly higher expression of miRNA-17 in the serum of patients with stage I NSCLC comparing to healthy individuals suggests that this microRNA may be a biomarker for diagnosis of early-stage NSCLC 48 . In the light of our results and literature data, a suggestion that the tumourigenic or tumour-suppressive functions of miR-17-5p might depend on the cellular context, that is, on the model system used, cell type or cancer stage seems to be right 39,42 .
A possibility of using of miR-17 as potential diagnostic tool in lung carcinogenesis has been discussed. The results of our study have shown that the level of miR-17 expression varies depending on the NSCLC histopathological subtype. It could speak in favour for a potential diagnostic function of miR-17 in NSCLC subtype differentiation. The results of others also support such a role 44,[49][50][51] . Interesting is that we obtained opposite results in cancerous and non-cancerous tissue for the both subtypes we examined. Such a change in the expression pattern of miRNA-17 may result from the high heterogeneity of tumour cells within the histological subtypes 52 . It may also be related to the fact that histopathological assessment does not always allow the detection of small clusters and individual tumour cells despite the finding of a negative histological margin, and transcript level dysregulation may be a sign of the ongoing process of neoplastic transformation in the margins surrounding the tumour 53 .
Comparative analyses between AC and SCC revealed upregulation of miR-17 in AC not only in solid tumour 44,50 , but also in plasma of NSCLC patients 45 . Although in our study a significant increase in the expression of miR-17 in the group of patients with AC was only observed in the tissue surrounding the tumour, the twofold change in the level of expression of miR-17 between AC and SCC, both in non-cancerous samples and tumour cells should be emphasized. This result provides evidence that miR-17 expression analysis could be useful as a support tool in NSCLC histopathological differential diagnosis. It remains to assume that, in accordance with previous reports 50,51 , SCC and AC employ different molecular pathways during their development and/ or progression. There is therefore a need for separate studies evaluating the biological effect of miR-17 on these two major NSCLC subtypes. www.nature.com/scientificreports/ Based on the assumption that PPARδ may be a molecular target for miR-17, analogously to PPARα 38,54 we have evaluated the correlation between the mRNA level of miR-17 and PPARδ. Numerous reports indicate that miRNAs directly target PPARs' mRNA or indirectly affect their expression [55][56][57] . To the best of our knowledge, so far only two articles have shown an increased expression of nuclear PPARβ/δ with simultaneous activation of miR-17-5p, which correlated with a decrease in inflammation and apoptosis of neurons and the molecular mechanism has been linked to the PPAR-β/δ/miR-17/TXNIP/NLRP3 signaling pathway 3,18 . However, the mechanism of this regulation (modulating PPARδ expression / activity by miRNA-17) in the context of oncogenesis has not been studied. Perhaps miR-17 indirectly affects PPAR-δ expression / activity by targeting PPARs-associated cofactors and repressors, thus providing a further level of complexity in these regulatory mechanisms that we have not studied.

Conclusions
In summary, due to the fact that we did not confirm the mutual correlation between miRNA-17 and PPARδ neither in the tumour tissue margins nor in NSCLC, we consider miR-17 and PPARδ as two independent molecular factors. The observed change in expression of miR-17 already at the margin of the tumour suggests that this miRNA may regulate basic biological processes (probably by enhancing cell proliferation) and therefore possibly plays an oncogenic role in the development of lung cancer. Our results indicate the potential role of the studied miRNA in the differentiation of NSCLC histopathological subtypes. Involvement of PPARδ in lung cancer biology is also undeniable, and the significant differences in expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer recognition. However, further research is required to verify the results.
Strengths and weaknesses of the study. The strengths of this study are: • the prospective design; • inclusion of NSCLC patients who have not been treated with potentially mutagenic chemotherapy or radiation therapy prior to surgery; • analysis in the most common NSCLC subtypes: AC and SCC; • the novelty aspect in the assessment of miR-17 and PPARδ co-expression in NSCLC; • analysis of PPARδ and miRNA-17 expression in both cancer lesion and macroscopically unchanged lung tissue (from the surgical margin).
The weaknesses of this study are: • small size of the research group; • lack of observation of patients with NSCLC to assess the concordance of miR-17 levels in blood and tissue; • lack of a validation set to confirm the obtained results in the ROC curve analysis.